Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.

10.Cronan JE, Jr. 2001. The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the “thumb” structure is essential and that the domain functions as a dimer. J Biol Chem276:37355–37364. [PubMed][CrossRef]

89.Barker DF, Campbell AM. 1981. Genetic and biochemical characterization of the birA gene and its product: evidence for a direct role of biotin holoenzyme synthetase in repression of the biotin operon in Escherichia coli. J Mol Biol146:469–492. [PubMed]

192.Brocklehurst SM, Perham RN. 1993. Prediction of the three-dimensional structures of the biotinylated domain from yeast pyruvate carboxylase and of the lipoylated H-protein from the pea leaf glycine cleavage system: a new automated method for the prediction of protein tertiary structure. Protein Sci2:626–639. [PubMed][CrossRef]

Two vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as “swinging arms” that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time. The late steps of biotin synthesis, those involved in assembling the fused rings, were well described biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like “arm” of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise, and the BioH esterase is responsible for its removal. In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl acyl carrier protein of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyltransferase followed by sulfur insertion at carbons C-6 and C-8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized, and, thus, there is no transcriptional control of the synthetic genes. In contrast, transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA, a dual-function protein that both represses biotin operon transcription and ligates biotin to its cognate proteins.

Structures of biotin, lipoic acid, n-octanoic acid, and the reduced form of lipoic acid, dihydrolipoic acid. (A) All biotin carbon atoms are numbered, as are the relevant carbons of the other molecules. (B) Stereochemistry of biotin and lipoic acid showing that both molecules have nonplanar structures. The lipoic acid dithiolane ring would emerge from and protrude below the plane of the page, whereas biotin has a chair structure (the viewer is looking at the back of the chair). Note that lipoic acid structure is rotated relative to that in panel A to conform with the Cahn-Ingold-Prelog rules, and, since biotin has three chiral centers, the hydrogen atoms attached to carbon atoms 7 and 10 can be depicted as either above or below the plane of the page depending on the chiral center chosen as primary (the ring centers were chosen in this depiction). For simplicity, the stereochemistry will not be given (except as relevant) in the subsequent figures of this review. doi:10.1128/ecosalplus.ESP-0001-2012.f1

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Figure 1

Structures of biotin, lipoic acid, n-octanoic acid, and the reduced form of lipoic acid, dihydrolipoic acid. (A) All biotin carbon atoms are numbered, as are the relevant carbons of the other molecules. (B) Stereochemistry of biotin and lipoic acid showing that both molecules have nonplanar structures. The lipoic acid dithiolane ring would emerge from and protrude below the plane of the page, whereas biotin has a chair structure (the viewer is looking at the back of the chair). Note that lipoic acid structure is rotated relative to that in panel A to conform with the Cahn-Ingold-Prelog rules, and, since biotin has three chiral centers, the hydrogen atoms attached to carbon atoms 7 and 10 can be depicted as either above or below the plane of the page depending on the chiral center chosen as primary (the ring centers were chosen in this depiction). For simplicity, the stereochemistry will not be given (except as relevant) in the subsequent figures of this review. doi:10.1128/ecosalplus.ESP-0001-2012.f1

The current model of the BioB reaction. For simplicity, only DTB carbon atoms 6, 7, 9, and 10 (Fig. 1) are shown, of which only carbons 6 and 9 are labeled. The reaction is shown as proceeding with the initial attack on C-9 because a derivative of DTB carrying a thiol group on C-9 has been shown to be converted to biotin both in vitro and in vivo (56, 260) and the crystal structure (24) shows C-9 in an appropriate position for the primary sulfur insertion. doi:10.1128/ecosalplus.ESP-0001-2012.f4

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Figure 4

The current model of the BioB reaction. For simplicity, only DTB carbon atoms 6, 7, 9, and 10 (Fig. 1) are shown, of which only carbons 6 and 9 are labeled. The reaction is shown as proceeding with the initial attack on C-9 because a derivative of DTB carrying a thiol group on C-9 has been shown to be converted to biotin both in vitro and in vivo (56, 260) and the crystal structure (24) shows C-9 in an appropriate position for the primary sulfur insertion. doi:10.1128/ecosalplus.ESP-0001-2012.f4

The biotin regulatory system of E. coli. BirA is represented by green ovals, biotin by black circles, the AMP moiety by red pentagons, AccB by dark blue ovals, and AccC by light blue crescents. The arrows denote transcription from the leftward and rightward bio promoters. (A to C) BirA switches from the biotin ligation function to the repressor function in response to the intracellular biotin requirement, which is monitored by the level of unbiotinylated AccB. If the levels of unbiotinylated AccB are high, the protein functions as a biotin ligase. Once the unbiotinylated AccB has been converted to the biotinylated form, the bio-AMP is no longer consumed and remains bound to BirA. This liganded form of BirA accumulates to levels sufficiently high to form dimers that fully occupy the bio operator, resulting in transcriptional repression of the biotin biosynthetic genes. (D) Overproduction of AccC ties up unbiotinylated AccB into a complex that is a poor biotinylation substrate. Therefore, high levels of the liganded form of BirA accumulate, resulting in repression of bio operon transcription. doi:10.1128/ecosalplus.ESP-0001-2012.f5

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Figure 5

The biotin regulatory system of E. coli. BirA is represented by green ovals, biotin by black circles, the AMP moiety by red pentagons, AccB by dark blue ovals, and AccC by light blue crescents. The arrows denote transcription from the leftward and rightward bio promoters. (A to C) BirA switches from the biotin ligation function to the repressor function in response to the intracellular biotin requirement, which is monitored by the level of unbiotinylated AccB. If the levels of unbiotinylated AccB are high, the protein functions as a biotin ligase. Once the unbiotinylated AccB has been converted to the biotinylated form, the bio-AMP is no longer consumed and remains bound to BirA. This liganded form of BirA accumulates to levels sufficiently high to form dimers that fully occupy the bio operator, resulting in transcriptional repression of the biotin biosynthetic genes. (D) Overproduction of AccC ties up unbiotinylated AccB into a complex that is a poor biotinylation substrate. Therefore, high levels of the liganded form of BirA accumulate, resulting in repression of bio operon transcription. doi:10.1128/ecosalplus.ESP-0001-2012.f5

The structure of BirA (A) and the sequence of the bio operon operator/promoter region (B). (A) The BirA structure is that of the protein liganded with a bio-AMP analogue (which for simplicity was omitted) (235). (B) The boxed region is the operator to which a BirA dimer binds. PA and PB are the promoters of the bioA and bioBFCD transcriptional units, respectively. The −10 and −35 promoter regions are denoted by underlines. doi:10.1128/ecosalplus.ESP-0001-2012.f6

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Figure 6

The structure of BirA (A) and the sequence of the bio operon operator/promoter region (B). (A) The BirA structure is that of the protein liganded with a bio-AMP analogue (which for simplicity was omitted) (235). (B) The boxed region is the operator to which a BirA dimer binds. PA and PB are the promoters of the bioA and bioBFCD transcriptional units, respectively. The −10 and −35 promoter regions are denoted by underlines. doi:10.1128/ecosalplus.ESP-0001-2012.f6

The BirA reaction. This is the general reaction of biotin protein ligases (38). The lipoic acid ligase LplA has the same reaction mechanism given substitution of lipoic acid for biotin. doi:10.1128/ecosalplus.ESP-0001-2012.f7

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Figure 7

The BirA reaction. This is the general reaction of biotin protein ligases (38). The lipoic acid ligase LplA has the same reaction mechanism given substitution of lipoic acid for biotin. doi:10.1128/ecosalplus.ESP-0001-2012.f7

Overview of the LipB reaction. The thioester linkage of octanoic acid attached to the thiol of the 4′-phosphopantheteine group of ACP (the product of fatty acid synthesis) is attacked by the ε-amino group of the target lysine of a lipoyl domain, resulting in the modified protein plus the free thiol form of ACP. The enzyme also uses lipoyl-ACP, although this is thought to be of no physiological importance. The reaction proceeds through an octanoyl-LipB acyl enzyme intermediate (not shown). doi:10.1128/ecosalplus.ESP-0001-2012.f9

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Figure 9

Overview of the LipB reaction. The thioester linkage of octanoic acid attached to the thiol of the 4′-phosphopantheteine group of ACP (the product of fatty acid synthesis) is attacked by the ε-amino group of the target lysine of a lipoyl domain, resulting in the modified protein plus the free thiol form of ACP. The enzyme also uses lipoyl-ACP, although this is thought to be of no physiological importance. The reaction proceeds through an octanoyl-LipB acyl enzyme intermediate (not shown). doi:10.1128/ecosalplus.ESP-0001-2012.f9

Current model for lipoic acid synthesis and utilization in E. coli. The rounded rectangle denotes an E. coli cell. Exogenous lipoic acid or octanoic acid enters by diffusion and is attached to the 2-oxoacid lipoyl domains and to H protein by LplA. The domains modified with exogenously derived octanoate can be converted to lipoyl domains by LipA, although this is probably not a reaction of physiological significance, because high levels of octanoic acid are required for significant modification by this route. In contrast, LplA-catalyzed attachment of lipoate is very efficient and provides a salvage or scavenging pathway for utilization of exogenous lipoic acid and seems likely to be physiologically significant. The major (and probably sole) route of lipoic acid synthesis is LipB-catalyzed transfer of octanoate from octanoyl-ACP to the 2-oxoacid lipoyl domains and H protein, followed by LipA-catalyzed sulfur insertion to give lipoate. doi:10.1128/ecosalplus.ESP-0001-2012.f10

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Figure 10

Current model for lipoic acid synthesis and utilization in E. coli. The rounded rectangle denotes an E. coli cell. Exogenous lipoic acid or octanoic acid enters by diffusion and is attached to the 2-oxoacid lipoyl domains and to H protein by LplA. The domains modified with exogenously derived octanoate can be converted to lipoyl domains by LipA, although this is probably not a reaction of physiological significance, because high levels of octanoic acid are required for significant modification by this route. In contrast, LplA-catalyzed attachment of lipoate is very efficient and provides a salvage or scavenging pathway for utilization of exogenous lipoic acid and seems likely to be physiologically significant. The major (and probably sole) route of lipoic acid synthesis is LipB-catalyzed transfer of octanoate from octanoyl-ACP to the 2-oxoacid lipoyl domains and H protein, followed by LipA-catalyzed sulfur insertion to give lipoate. doi:10.1128/ecosalplus.ESP-0001-2012.f10

Lipoate synthesis proceeds by sulfur insertion into octanoyl domain. The bypass pathway accounting for the growth of lipB mutants on octanoate is shown in the upper-right cartoon. The experimental protocol scheme and mass spectral data for testing the pathway are also shown. In the left cartoon, octanoylation of the lipoyl domain by endogenously synthesized octanoyl moieties is blocked by a lipB mutation, and the cells use LplA and exogenously supplied d15 octanoate to octanoylate the domain. LipA is also blocked so deuterated lipoylated domain is not made. Following accumulation of the deuterated octanoyl domain, LipA function is restored (right cartoon). Following incubation to allow lipoate synthesis, the cells were harvested and the modified domains were purified and then analyzed by electrospray mass spectrometry. Note the accumulation of deuterated lipoylated domain (D-Lip) in the right hand spectrum and that the mass change between deuterated octanoylated domain (D-C8) and D-Lip is 60 mass units, indicating the loss of two deuterons and the gain of two sulfur atoms. For details, see reference 201. doi:10.1128/ecosalplus.ESP-0001-2012.f11

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Figure 11

Lipoate synthesis proceeds by sulfur insertion into octanoyl domain. The bypass pathway accounting for the growth of lipB mutants on octanoate is shown in the upper-right cartoon. The experimental protocol scheme and mass spectral data for testing the pathway are also shown. In the left cartoon, octanoylation of the lipoyl domain by endogenously synthesized octanoyl moieties is blocked by a lipB mutation, and the cells use LplA and exogenously supplied d15 octanoate to octanoylate the domain. LipA is also blocked so deuterated lipoylated domain is not made. Following accumulation of the deuterated octanoyl domain, LipA function is restored (right cartoon). Following incubation to allow lipoate synthesis, the cells were harvested and the modified domains were purified and then analyzed by electrospray mass spectrometry. Note the accumulation of deuterated lipoylated domain (D-Lip) in the right hand spectrum and that the mass change between deuterated octanoylated domain (D-C8) and D-Lip is 60 mass units, indicating the loss of two deuterons and the gain of two sulfur atoms. For details, see reference 201. doi:10.1128/ecosalplus.ESP-0001-2012.f11

A current model of the lipoate synthase (LipA) reaction (202, 203, 240). The canonical SAM radical [4Fe-4S] cluster of LipA reduces SAM to generate the deoxyadenosine radical (5′-Ado) as seen previously in the BioB reaction (Fig. 4). The radical then removes a hydrogen atom from the C-6 methylene of the octanoate moiety of an octanoyl domain (Oct-E2 on the figure) (229) to give a carbon radical that then attacks the lipoyl synthase-specific [4Fe-4S] cluster and abstracts a reduced sulfur atom. This process is repeated at the methyl carbon (C-8) to give lipoyl domain (Lip-E2, probably as the dihydrolipoyl form due to the strongly reducing conditions under which the reaction proceeds). doi:10.1128/ecosalplus.ESP-0001-2012.f12

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Figure 12

A current model of the lipoate synthase (LipA) reaction (202, 203, 240). The canonical SAM radical [4Fe-4S] cluster of LipA reduces SAM to generate the deoxyadenosine radical (5′-Ado) as seen previously in the BioB reaction (Fig. 4). The radical then removes a hydrogen atom from the C-6 methylene of the octanoate moiety of an octanoyl domain (Oct-E2 on the figure) (229) to give a carbon radical that then attacks the lipoyl synthase-specific [4Fe-4S] cluster and abstracts a reduced sulfur atom. This process is repeated at the methyl carbon (C-8) to give lipoyl domain (Lip-E2, probably as the dihydrolipoyl form due to the strongly reducing conditions under which the reaction proceeds). doi:10.1128/ecosalplus.ESP-0001-2012.f12